禽流感病毒与新城疫病毒多靶标核酸质谱检测方法研究及应用

doi:10.11843/j.issn.0366-6964.2025.03.037

Abstract

Abstract:

Avian influenza (AI) and Newcastle disease (ND) are the two most threatening viral infectious diseases in the global poultry industry. Rapid and accurate pathogen detection and typing are of vital importance for the early control and transmission blocking of these two animal diseases. (Method) In this study, a high-throughput nucleic acid mass spectrometry method for detection of AIV and NDV was developed by combining micro multiplex RT-PCR, single base extension and MALDI-TOF mass spectrometry, and the sensitivity, specificity and repeatability of the method were evaluated. The results showed that the established method could detect AIV (type A), subtype H5, subtype H7, subtype H9, NDV and its velogenic/mesogenic strains synchronously. The analytical sensitivity of targets was 8.82-15.40 copies·μL^-1, and there was no cross-reaction with other avian pathogen nucleic acid. The reproducibility of 6 mixed targets at 12 copies·μL^-1 was above 75%. The detection results of 179 samples (including swab samples and tissue organs) collected in laboratory showed that the coincidence rate of universal target of AIV was 98.9%, and the other targets were 100.0% compared with real-time RT-PCR. So a new high-throughput alternative method is provided for differential detection of AIV, NDV, and their important subtypes/strains synchronously in this study.

Key words: avian influenza virus, Newcastle disease virus, multi-target, nucleic acid mass spectrometry

CLC Number:

S851.34

GAO Zhiqiang, LAI Ping'an, SONG Yueqian, CHONG Yan, GUO Youran, BAI Zilong, GUO Huimin, WANG Lin, PU Jing, SHI Xiju, REN Tong, ZHAO Xiangpeng. Studies and Application of Multi-target Nucleic Acid Mass Spectrometry Detection Method for Avian Influenza/Newcastle Disease Virus[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(3): 1386-1395.

Figures/Tables 8

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References 11

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靶标 Target	基因 Gene	扩增引物(5′→3′) Amplification primers	延伸引物(5′→3′) Extension primers	延伸引物质量/u Mass of unextended primers	延伸产物质量/u Mass of extended primers
AIV	M	F: ACGTTGGATGCCRATCYTGTCACCTCTGACTAA	TCACTGGGCACGGTGA	4 922.24	5 209.44
		R: ACGTTGGATGCGTCTACGCTGCAGTCCTC
H5	HA	F1: ACGTTGGATGATTTAYTCAACAGTGGCGRGTT	GCCTAGCACTGGCAATC	5 468.64	5 715.84(C)
		F2: ACGTTGGATGATTTAYTCAACAGTGGCGAGCT			5 795.84(T)
		R1: CTACAATCTGAACTCACAAGTTTARATGC	TCCCTAGTGCTGGCAATCA	5 763.84	6 011.04(C)
		R2: CTACAATCTGAACTCACAGCTCTARATGC			6 091.04(T)
			TGGATGTGCTCCAATGG	5 241.44	5 512.64(A)
					5 528.64(G)
H7	HA	F: ACGTTGGATGAAAATAGAATACAGATWRACCCRGT	CTTCGGGGCATCATGTTT	5 496.64	5 823.84(T)
		R: ACGTTGGATGGTGCACYGCATGTTTCC			5 743.84(C)
H9	HA	F: CGGATAACAATTTCACACAGGATGGGGTTTGCTGCC	GACATGGCCCAGAA	4 281.84	4 553.04(T)
		R: ACGTTGGATGAATTATATACAAATGTTGCAYCTG			4 529.04(G)
					4 609.04(A)
			GGTGTTGGACATAGCCCAGAA	6 495.24	6 766.44(T)
					6 742.44(G)
					6 822.44(A)
NDV	M	F: ACGTTGGATGTATGRTGRTAACWTGCAAGAAGAG	AAGGTGCCTGCACTA	4 577.04	4 824.44(G)
		R: ACGTTGGATGGCTTTCACRTGCTTVACTG			4 904.24(A)
NDV	F	F: ACGTTGGATGTCCGGAGGATACAAGRGTCYG	CCGGAGGAAGGAGACAGA	5 631.64	5 918.84(G)
中强毒株		R: ACGTTGGATGAGCTGTTGCRACCCCAAG			5 902.84(A)
NDV			CGGAGGAAGGAGACAAA	5 326.44	5 613.64(G)
					5 597.64(A)
mesogenic strains			CCGGAGGAAGGAGGCAGA	5 976.84	6 264.04(G)
					6 248.04(A)
			GTCTGGAGGAAGGAGACGGA	6 296.04	6 583.24(G)
					6 567.24(A)
IC	β-actin	F: ACGTTGGATGCCTTCAACACCCCTGCCATG	GTGCTGTCCCTGTACGCCTCTGG	6 998.60	7 325.8(T)
		R: ACGTTGGATGGTCCATCACGATRCCRGTGGT			7 245.80(C)
			CTGCTGTCCCTGTATGCCTCTGG	6 973.60	7 300.80(T)
					7 220.80(C)
			CTGCTGTCTCTGTACGCCTCTGG	6 973.60	7 300.80(T)
					7 220.80(C)

混合RNA浓度/(copies·μL^-1) Mixed RNA concentration	靶标名称 Target name	检测结果(Pos/Total) Detection result	阳性率/% Positive rate
50	AIV-M	8/8	100
	H5-HA	8/8	100
	H7-HA	8/8	100
	H9-HA	8/8	100
	NDV-M	8/8	100
	NDV-VF	8/8	100
25	AIV-M	8/8	100
	H5-HA	8/8	100
	H7-HA	8/8	100
	H9-HA	8/8	100
	NDV-M	8/8	100
	NDV-VF	8/8	100
12	AIV-M	7/8	87.5
	H5-HA	8/8	100
	H7-HA	7/8	87.5
	H9-HA	6/8	75
	NDV-M	7/8	87.5
	NDV-VF	8/8	100

检测靶标 Test target	数量 Number	检测结果 Test results				符合率/% Coincidence	Kappa
检测靶标 Test target	数量 Number	RRT-PCR(—)/MS(—)	RRT-PCR(+)/MS(+)	RRT-PCR(—)/MS(+)	RRT-PCR(+)/MS(—)	符合率/% Coincidence	Kappa
AIV 通用	179	122	55	0	2	98.9	0.974
AIV general
H5	179	167	12	0	0	100	1.0
H7	179	175	4	0	0	100	1.0
H9	179	161	18	0	0	100	1.0
NDV通用	179	174	5	0	0	100	1.0
NDV general
NDV中强毒株	179	174	5	0	0	100	1.0
NDV mesogenic strains

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Studies and Application of Multi-target Nucleic Acid Mass Spectrometry Detection Method for Avian Influenza/Newcastle Disease Virus

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